The miR-641-STIM1 and SATB1 axes play important roles in the regulation of the Th17/Treg balance in ITP

Immune thrombocytopenia (ITP) is an autoimmune disease caused by T-cell dysfunction. Recently, several studies have shown that a disturbed Th17/Treg balance contributes to the development of ITP. MicroRNAs (miRNAs) are small noncoding RNA moleculesthat posttranscriptionally regulate gene expression. Emerging evidences have demonstrated that miRNAs play an important role in regulating the Th17/Treg balance. In the present study, we found that miR-641 was upregulated in ITP patients. In primary T cells, overexpression of miR-641 could cause downregulation of its target genes STIM1 and SATB1, thus inducing a Th17 (upregulated)/Treg (downregulated) imbalance. Inhibition of miR-641 by a miR-641 sponge in primary T cells of ITP patients or by antagomiR-641 in an ITP murine model could cause upregulation of STIM1 and SATB1, thus restoring Th17/Treg homeostasis. These results suggested that the miR-641-STIM/SATB1 axis plays an important role in regulating the Th17/Treg balance in ITP.


Pan-T-cell isolation
Pan-T cells were isolated using a Pan-T-cell isolation kit, human (Miltenyi Biotec, 130-096-535) or a Pan-T-cell isolation kit, mouse (Miltenyi Biotec, 130-095-130) following the manufacturer's instructions.In brief, PBM-NCs from peripheral blood were collected using Ficoll Paque Plus (M19059, GE Healthcare).The cells were counted, and the amount of reagents used for subsequent experiments was calculated based on the cell count.The cells were centrifuged at 1200 rpm for 5 min and resuspended in 40 µl of buffer (AutoMACS Running buffer:AutoMACS Risining solution = 1:20) per 10^7 cells.The mixture was transferred to 1.5 ml EP tubes.Then, 10 µl of Pan-T-cell Biotin-Antibody Cocktail was added, mixed well and incubated at 2-8 °C for 5 min.Thirty microliters of buffer was added per 10^7 cells.Ten microliters of Pan-T-cell MicroBead Cocktail was added to each unit, and the mixture was mixed well and incubated at 2-8 °C for 10 min.The LS column and magnetic field were prepared, the column was rinsed with 3 ml of buffer, and the cell suspension was applied to the column.The flow-throughs containing unlabeled cells, representing the enriched T cells, were collected.

qRT-PCR (miRNA and mRNA)
For miRNA qRT-PCR, the miDETECTATrackTM miRNA qRT-PCR Starter Kit (Guangzhou RiboBio Co., Ltd., China, Inc.) was used to reverse transcribe the total RNA to cDNA andamplify the RT product according to the manufacturer'sinstructions.Theprimer sequences were synthesized by Guangzhou RiboBio Co., Ltd., China, andare summarized in the Supplementary Table .For target gene coding mRNA primer design, the PICK PRIMERS tool in the nucleic acid module of NCBI (https:// www.ncbi.nlm.nih.gov/ nucco re) was used.qRT-PCR

Plasmid construction for miR-641 OE or miR-641 sponge
The miR-641 overexpression and miR-641sponge plasmids were constructed (HANBIO Biotechnology (Shanghai) Co.) (see Table S2, S3 for plasmid structure and sequence details), and polybrene was used for the viral transfection enhancement solution.For primary T-cell transfection, the MOI was set to 100, and for cell line transfection, the MOI was set to 50.Primary T-cell transfection was performed in 6-well plates, while cell lines were used in 24-well plates.
The percentages of Treg and Th17 cells were then measured using flow cytometry (BD FACS Canto II,BD Biosciences) and analyzed using FlowJo V10 (FlowJo LLC, BD Biosciences).
The ITP murine model was induced by intraperitoneal injection of the antiplatelet antibody MWReg30 using a micro-osmotic pump (Alzet micro-osmotic pump, Model 1002; Alza, Palo Alto, CA).The micro-osmotic pump was filled with 100 μl of sterile saline containing MWReg30 (82.5 μg/ml) and bovine serum albumin (1.5 mg/ ml) and inserted into the peritoneal cavity of the mice.MWReg30 was released at a rate of 0.5 μl/h for 8 days 27,28 .Mice in the AntagomiR-641 group were injected with the micrOFF hsa-miR-641 antagomir (miR30003311-4, Guangzhou RiboBio Co. Ltd.China) via the tail vein 4 days after the construction of the ITP murine model.The micrOFF™ miRNA antagomir is a specially chemically modified miRNA antagonist that inhibits the action of miRNAs by competitively binding to mature miRNAs in vivo, preventing complementary pairing of miRNAs with their target mRNAs.The miRNA antagomir is the reverse complementary sequence of the mature miRNA strand to the whole strand.They are modified by methylation, with two-and four-base sulfation modifications at the 5' and 3' ends, respectively, and a high-affinity cholesterol modification at the 3' end 29 .All mice were sacrificed on Day 8, and their bone marrow and peripheral blood were collected for further investigations (Fig. 5A).

Cytokine assay
3 mice in each group were randomly selected for Cytokine Assay.Merck Millipore liquid phase microarray assays were used to detect plasma cytokines.The microspheres are color coded internally using a variety of fluorescent dyes.The precise concentration of these dyes allows the creation of clear colored bead sets of 500 nonmagnetic microspheres (5.6 µm) or 80 magnetic polystyrene microspheres (6.45 µm), each coated with a specific capture antibody.After the beads were captured and analyzed in the test sample, biotinylated detection antibodies were introduced.The reaction mixture was then incubated with the reporter molecule streptavidin PE conjugate to complete the reaction on the surface of each microsphere.Each microsphere was identified, and its bioassay was quantified based on the fluorescent reporter signal.Assays were performed with Luminex® 200TM software.The median fluorescence intensity (MFI) data were saved and analyzed, and the concentration of the protein to be detected in the sample was calculated using a 5-parameter (5pl) fitted curve.

Giemsa staining
3 mice in each group were randomly selected for Giemsa Staining.Wright-Giemsa staining was conducted using Wright-Giemsa staining solution (BA4017A, BASO, China) according to the manufacturer's instructions.In brief, bone marrow smears were prepared and fixed with absolute methanol for 2 min.The cells were stained with Wright-Giemsa Stain Solution for 1 min at room temperature.After staining, the slides were rinsed in PBS for 5 min, rinsed in running deionized water (5-6 immersions), and then allowed to air dry.Slides were observed and photographedwith an Axio Scope A1 microscope (Microscopes, Software & Imaging Solutions ZEISS, Germany).The cell types were identified by a pathologist.Giemsa Staining underwent biological repilication (n = 3).

Platelet count
3 mice in each group were randomly selected for Platelet count.Platelet counts were conducted using a Sysmex XN-1000B1 automated hematology analyzer (Sysmex Europe GmbH, Germany).EDTA-treated peripheral blood was collected from the mice in each group and analyzed following the manufacturer's instructions.Platelet count analysis underwent biological replication (n = 3).The platelet count was statistically analyzed.

Statistical analysis
Quantitative data are presented as the mean ± standard error ofthe mean (SEM).Comparisons between groupswere made with one-way ANOVA or the Mann-Whitney U test.Differences were considered significant at *P < 0.05 or ***P < 0.001.

Statement on guideline and consent
The study was approved by the guidelines institutional ethics committee of The Second Xiangya Hospital, Central South University.All methods were performed in accordance with the guidelines institutional ethics committee of The Second Xiangya Hospital, Central South University.The animal experiment were performed strictly according to ARRIVE guidelines.Mice were anesthetized or sacrificed according to American Veterinary Medical Association guidelines.All the animal study are approved by the Animal Ethic of The Second Xiangya Hospital, Central South University.www.nature.com/scientificreports/

Ethical approval and consent to participate
Patient samples were collected after informed written consent was obtained accordance with the Declaration of Helsinki.This study was approved by the institutional ethics committee of The Second Xiangya Hospital, Central South University.

Statement
This study was approved by the institutional ethics committee of The Second Xiangya Hospital, Central South University.The study is reported in accordance with ARRIVE guidelines.

Results
1. Differentially expressed miR-641 and its potential target mRNAs qRT-PCR was performed to investigate the expression of miR-641 and its target genes (SATB1 and STIM1) in the peripheral T cells of ITP patients and in normal candidates (NCs) (Table 1).The results showed that miR-641 expression was upregulated in ITP patients compared with that in NCs (p < 0.05) (Fig. 1A).
2. Identifying the potential target genes of miR-641 in the T cells of ITP patients The Mirdb database (http:// mirdb.org/ datab ase) was used to predict the potential target genes of hsa-miR-641 in both humans and mice.By cross-checking published articles describing important genes correlated with T-cell regulation, 2 potential target genes (SATB1 and STIM1) were selected for further investigation.The results of qRT-PCR analysis showed that the expression of both SATB1 (p < 0.001) and STIM1 (p < 0.001) was significantly downregulated in the T cells of ITP patients compared with those of NCs (Fig. 1B,C).Dual-luciferase analysis was further performed to investigate the interaction between miR-641 and its potential target genes (SATB1 and STIM1).We observed a direct interaction of miR-641 with SATB1 and STIM1 (Fig. 2A,B,C).
293 T cells were transfected with lentivirus (miR-641) to overexpress miR-641 (Fig. 2D).The expression of SATB1 and STIM1 was analyzed by qRT-PCR (Fig. 2E,F), and the expression of their encoded proteins was analyzed by Western blotting (Fig. 2G).The results suggested that overexpression of miR-641 could lead to downregulation of the expression of SATB1 and STIM1 mRNA and their encoded proteins.
3. The overexpression of miR-641 in T cells isolated from normal candidates leads to an imbalance of Th17/ Treg cells.
Cytokine assays revealed that IL17A promoted Th17 differentiation and was downregulated in the antago-miR-641 group compared with the ITP group but did not reach the level of the MOCK group (Fig. 7A).Compared with that in the ITP group, IFNγ downregulated Th17 and upregulated Treg cell numbers and was upregulated in the AntagomiR-641 group, and IFNγ expression was upregulated in the AntagomiR-641 group compared with the MOCK group (Fig. 7B).IL6, a regulator of the Treg/Th17 balance, inhibits TGFβ-induced Treg differentiation and induces Th17 differentiation.This cytokine was downregulated in the AntagomiR-641 group compared to the ITP group and was comparable to that in the MOCK group (Fig. 7C).Th17 cells promoted the expression of MCP1, which was significantly downregulated in the AntagomiR-641 group compared with the ITP group and significantly inhibited compared with the MOCK group (Fig. 7D).IL4 inhibited Th17 differentiation, a cytokine that was upregulated in the AntagomiR-641 group compared to the ITP group but did not reach the expression level of the MOCK group (Fig. 7E).IL-2 promoted Treg differentiation, and this cytokine was upregulated in the AntagomiR-641 group compared to the ITP group, and the levels were comparable to those in the MOCK group (Fig. 7F).IL13 is a Th2-type cytokine that promotes Treg differentiation; this cytokine was upregulated in the AntagomiR-641 group compared to the ITP group, and its levels were comparable to those in the MOCK group (Fig. 7G) .Tregs secrete IL-10, which inhibits T-cell activation.This cytokine was upregulated in the AntagomiR-641 group compared to the ITP group and was comparable to that in the MOCK group (Fig. 7H).TNFa reportedly increases the number of Tregs and upregulates the expression of Foxp3 in patients with IBD.The results of this study revealed no significant difference in the expression of this cytokine between the AntagomiR-641 group and the ITP group or between the AntagomiR-641 group and the MOCK group, but there was no significant difference (Fig. 7I).
According to Deka et al., megakaryocytes in ITP patients have a greater nuclear/cytoplasmic ratio, lower nuclear roundness factor and lower nuclear contour ratio.Cellular circularity and compactness were significantly different between ITP patients and non-ITP patients, suggesting that there were fewer megakaryocytes in ITP patients than in non-ITP patients 30 .In our study, bone marrow smears following Giemsa staining were used to observe the morphology of megakaryocytes in each group.Compared with those in the MOCK group, the megakaryocytes in the ITP group had a greater nuclear/cytoplasmic ratio, were less circular and increased in cell size; however, compared with those in the ITP group, the megakaryocytes in the AntagomiR-641 group had a decrease in cell size and more circular morphology but still had a greater nuclear/cytoplasmic ratio (Fig. 8A,B,C).The platelet counts suggested that the amount of platelets in the peripheral blood of the ITP group was greatly decreased compared with that in the peripheral blood of the MOCK group, while it was increased in the Antago-miR 641 group (Fig. 8D).

Discussion
In the present study, we obtained the following findings.D,E) FACS analyzed the Th17 cells surface markers CD4 and IL-17A expression after miR641 over-expression (lentivirus transfection) in primary T cells from 3 normal candidates.(F) Statistic analysis of FACS results: CD4 + IL17A + Th17 cells were up-regulated in miR641 over-expression primary T cells from normal candidates (Red Column) comparing with vector only group (Blue Column).(G,H) FACS analyzed the Treg cells surface markers CD25 and Foxp3 expression after miR641 over-expression (lentivirus transfection) in primary T cells from 3 normal candidates.(I) Statistic analysis of FACS results: CD25 + Foxp3 + cells were up-regulated in miR641 over-expression primary T cells from 3 normal candidates (Red Column) comparing with vector only group (Blue Column).
1. miR-641 was upregulated in ITP patients.The interaction of miR-641 with SATB1 and STIM1 was observed by dual-luciferase reporter gene reporter assays.The overexpression of miR-641 in 293 T cells reduced SATB1 and STIM1 levels.2. The overexpression of miR-641 in human primary T cells in vitro resulted in the downregulation of SATB1 and STIM1 expression and could cause the upregulation of Th17 cells and the downregulation of Treg cells.3. Using a miR-641 sponge to block miR-641 expression in ITP patients' primary T cells resulted in the upregulation of SATB1 and STIM1 expression, the downregulation of Th17 cells, and the upregulation of Treg cells.4. In a murine ITP model, tail vein injection of antagomiR-641 downregulated peripheral miR-641 expression and upregulated SATB1 and STIM1 expression, thus restoring the Th17/Treg balance while also correspondingly regulating changes in peripheral plasma cytokine expression and increasing the PLT.

Progression of ITP treatment
ITP is a tissue-specific autoimmune disease.Current knowledge of this disease suggests that its development is closely related to the dysfunction of immune cells such as T cells, B cells, dendritic cells and macrophages.First-line treatments for ITP include steroids, intravenous immunoglobulin (IVIg) and/or Rh immune globulin (anti-D).Second-and third-line treatments include roxan, fostamatinib, thrombopoietin receptor agonists (TPO-RAs), and/or splenectomy.Treatment failure in ITP patients is usually associated with severe immune system dysfunction 31,32 .T-cell immune abnormalities play a key role in the development of ITP.Th1, Th2, Th17, Th22, Tfh and plasma IL-22, IL-17A, and IFN-gamma expression was upregulated in the plasma of ITP patients.CD4 + CD25 + Treg cells play an important role in maintaining peripheral immune tolerance and are significantly reduced in both the peripheral blood and bone marrow of ITP patients.Accumulating evidence emphasizes that targeting T cells might be a potential strategy for ITP treatment.Early in 2014, Ji and colleagues reported that haloguginone significantly recovered peripheral platelet counts in ITP mice by promoting Th1 cell differentiation and attenuating Th2 differentiation 4 .Many research groups have reported that restoring the Th17/Treg imbalance could achieve therapeutic benefits for ITP.Guo and colleagues reported that resveratrol couldplay a therapeutic role in ITP by restoring the Th17/Treg imbalance 9 .Indirubin regulates CD4+ T-cell homeostasis in ITP via the PD1/PTEN/AKT signaling pathway 12 .Dexamethasone modulates the polarization of Th17 cells to CD4+ T cells in patients with primary ITP 14 .Halofuginone has also been reported to exert a therapeutic effect by modulating T-cell function in a mouse model of ITP 33 .
In addition to traditional drug therapy, research into the role of noncoding RNAs in the pathogenesis of ITP has been intensively conducted in recent years.The long noncoding RNA MEG3 inhibits microRNA-125a-5p expression and induces an imbalance of Treg/Th17 cells in ITP 34 .Hua and colleagues reported that the expression of microRNAs in CD4 + cells contributes to Th17/Treg imbalance in ITP.They found that miR99a expression was lower in ITP patients, while miR-183-5p and miR-183-5p were more highly expressed in ITP patients 11 .Li et al. reported that miR-106p-5p induces an imbalance of Treg/Th17 cells in ITP through the NR4A3/Foxp3 pathway 21 .Decreasing lncRNA PVT1 causes Treg/Th17 imbalance via NOTCH signaling in ITP 20 .

Functions of miR-641
Previous studies have shown that miRNA641 is involved in regulating the progression of a variety of solid tumors by regulating the expression of its target genes.Intronic miRNA-641 controls its host gene pathway PI3K/AKT, and this relationship is dysfunctional in glioblastoma multiforme 35 .miR-641 Functions as a Tumor Suppressor by Targeting MDM2 in Human Lung Cancer 36 .The lncRNA TUSC8 inhibits the invasion and migration of cervical cancer cells via the miR-641/PTEN axis 37 .However, the role of miR-641 in the regulation of immune system function has not been reported.Lamya Garabet et al. reported the changes in the expression levels of circulating miRNAs in ITP patients before and after TPO-RA treatment 38 .The authors screened out six miR-NAs that showed significant changes, but our reported miR641 was not included.This could be due to different observation subjects: this study observed the differences in miRNAs between ITP patients and normal healthy

Conclusion
1.For the first time, miR-641 expression in T cells from ITP patients was described, and its interactions with the target genes SATB1 and STIM1 were screened and confirmed.2. miR-641-SATB11 and STIM1 were found to play important roles in Th17/Treg regulation in the peripheral blood T cells of normal individuals and ITP patients.3. Therefore, targeting the miR-641-SATB1/STIM1 axis might be a potential strategy for ITP therapy.

Figure 5 .
Figure 5. (A) Schema of in vivo experiments.(B) qRT-PCR analyzed miR641 expression in the peripheral blood from MOCK group (dark blue column), ITP group (Red column) and AntagomiR641group (light blue column).(C) qRT-PCR analyzed SATB1 expression in the peripheral blood from MOCK group (dark blue column), ITP group (Red column) and AntagomiR641group (light blue column).(D) qRT-PCR analyzed STIM1 expression in the peripheral blood from MOCK group (dark blue column), ITP group (Red column) and AntagomiR641group (light blue column).qRT-PCR analysis underwent biological replication (n = 3).

Table 1 .
ITP Patients and Normal Candidates.